Development of novel assays for lignin breakdown and identification of a new bacterial lignin degrading enzyme

Abstract

Two novel spectrophotometric assays have been developed for high throughput screening of microbial lignin degradation. The first assay involves fluorescently labelled lignin, which gives a time dependent increase in fluorescence with lignin degradation. The second involves nitrated lignin where an increase is seen at 430nm when lignin is broken down. These assays have allowed identification of a number of new strains of lignin degrading bacteria including R. jostii RHA1. They have also allowed the comparison of bacterial and fungal degraders and a study of lignin specificity. Bioinformatics has been used to identify two putative lignin degrading enzymes in R. jostii RHA1. Their role in lignin degradation was investigated by assaying of gene knockouts. This led to the detailed study of a new recombinant peroxidase, DypB, using lignin, lignocellulose and lignin dimer model substrates. This represents the first recombinant bacterial lignin degrading enzyme to be characterised. In addition methods for studying the products of lignin breakdown by HPLC, GC/MS and LC/MS have been developed. This has led to the identification of several low molecular weight aromatic products including ferulic acid. It has also been shown that the products released from lignin model compounds can be controlled by addition of the reductive enzyme diaphorase